Treating diabetes with genetically modified beta cells

ABSTRACT

Described herein are human transgenic beta cells expressing fugetactic levels of CXCL12 to a subject in need thereof. Also described herein are beta cells comprising a transgene comprising a nucleic acid sequence encoding CXCL12.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/146,980, which claims priority to U.S. Provisional Application Nos.62/567,604, filed Oct. 3, 2017; 62/568,117, filed Oct. 4, 2017;62/637,913, filed Mar. 2, 2018; 62/662,651, filed Apr. 25, 2018;62/694,634, filed Jul. 6, 2018; 62/696,603, filed Jul. 11, 2018;62/717,587, filed Aug. 10, 2018; 62/719,975, filed Aug. 20, 2018; and62/734,910, filed Sep. 21, 2018; each of which is incorporated byreference herein in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 25, 2018, isnamed 054610-501001US_SL.txt and is 8,646 bytes in size.

FIELD OF THE INVENTION

The invention is directed to genetically modified, human beta cells aswell as methods using such cells. The genetically modified (transgenic),human beta cells express a fugetactic amount of a fugetactic agentthereby imparting protection against human mononuclear immune cells. Inone embodiment, the fugetactic agent is, for example, CXCL12 or CXCL13.In one embodiment, the transgenic beta cells comprise a vector, whereinthe vector comprises a nucleic acid sequence encoding a fugetactic agentand preferably a human fugetactic agent. In one embodiment, thetransgenic beta cells are further modified to be senescent. Methods ofthis invention include use these cells to express insulin in ahyperglycemic environment including those found in diabetic patients, inparticular type I diabetic patients.

BACKGROUND OF THE INVENTION

Beta cells are responsible for producing insulin in the pancreas. Insubjects with type 1 diabetes (T1D), beta cells are attacked anddestroyed by the immune system and, as a result, subjects with T1Dcannot efficiently produce their own insulin. Cloning technique used tocreate pancreatic cells for type 1 diabetes, Diabetes. Co. Uk,www.diabetes.co.uk/news/2014/apr/cloning-technique-used-to-create-pancreatic-cells-for-type-1-diabetes-94233303.html(Apr. 29, 2014).

Type 2 diabetes (T2D) occurs when a subject's persistently high bloodsugar overwhelms the capacity of a subject's beta-cells to produceenough insulin to prevent hyperglycemia in the subject and leads tobeta-cell malfunction, de-differentiation, and death. Felicia W Pagliuca& Douglas A. Melton, How to make a functional β-cell,” Development 2013,140(12), 2472-2483.

Allogeneic beta cell transplantation, also known as islet celltransplantation, from a normal donor to a diabetic recipient has beenconsidered as a method of treating diabetes. However, infiltration ofmononuclear immune cells (T-cells, B-cells, and NK cells) results in thefailure of the beta cell transplantation. Cloning technique used tocreate pancreatic cells for type 1 diabetes, Diabetes. Co. Uk,<www.diabetes.co.uk/news/2014/apr/icloning-technique-used-to-create-pancreatic-cells-for-type-1-diabetes-94233303.html>(Apr.29, 2014); Alan H. Cruickshank & Emyr W Benbow, “Recurrence ofDiabetes,” Pathology of the Pancreas (3d ed. 1995); Felicia W Pagliuca &Emyr W Benbow, “Recurrence of Diabetes,” Pathology of the Pancreas (2ded. 1995); Felicia W Pagliuca & Douglas A. Melton, “How to make afunctional β-cell,: Development 2013, 140(12):2472-2483.

Current clinical practice is to transplant islets containing beta cellsinto the liver via the portal vein, with the rationale that the majorityof insulin released from the pancreas is utilized in the liver and thesite is easily accessible by a minimally invasive procedure. However,half of the beta cells die shortly after transplantation, and this isthought to be due to low oxygen tension, an active immune response, andhigh levels of toxins and drugs in the liver. In addition, the instantblood mediated inflammatory reaction (IBMIR) encapsulates transplantedislets in a fibrin clot and enhances the immune reaction against thegraft. Therefore, several alternative sites for transplantation havebeen tested including the intestine, kidney capsule, omentum, andsubcutaneous, which may be best for patient safety, but have not beenfully evaluated for systemic release of insulin.

To prevent their exposure to mononuclear immune cells, beta cells havebeen encapsulated in devices that have been reported to serve a dualfunction of isolating these cells from immune destruction and protectingthe host from the graft. Immune protection is required whennon-autologous cells (e.g., allogeneic or xenogeneic cells) are used fortransplantation, or if autologous cells are transplanted into anautoimmune environment, such as a Type 1 diabetic patient. This can beachieved by blocking the cellular response via physical isolation of thecells using semi-permeable membranes or scaffolds. This approach reducesthe need for immunosuppression and has recently been reviewed in detailelsewhere (Sakata et al, World J Gastrointest Pathophysiol. 2012;3:19-26; Qi, Adv Med. 2014; 2014:429710). Encapsulation also preventscells from escaping the location of the graft, and allows for removal,if needed. This is particularly relevant, as uncontrolleddifferentiation and growth, e.g. teratomas, have often been observed inmice grafted with stem cell-derived pancreatic precursors (Kroon et al,Nat Biotechnol. 2008; 26:443-452; Kelly et al, Nat Biotechnol. 2011;29:750-756.). Teratoma formation may be preventable by generating graftsconsisting of a pure population of mature beta cells, either by usingcell purification (Kelly et al, 2011) or by improving differentiationmethods. Encapsulation also prevents potential metastasis ifinsulinomas/teratomas are formed by the transplanted cells.

This approach requires efficient and reproducible micro-encapsulationprotocols, and some cells may die within the encapsulated materialwithout the possibility of being cleared or removed. Some reports haveshown micro-encapsulated beta cells can remain viable up to 6 monthsafter implantation (Orlando et al, Diabetes. 2014; 63:1433-1444). Thismeans that repeated surgeries are required to replace themicro-encapsulation devices when the device is no longer functional.

In view of the above, there is a long unmet need to develop technologythat effectively treats diabetes and, in particular, type 1 diabetes.

SUMMARY OF THE INVENTION

This invention is directed to transgenic, human beta cells as well astransgenic, senescent, human beta cells that express an effective amountof a fugetactic agent so as to render these cells resistant to humanimmune cells. Fugetactic agents are well known in the art including“CXCL12”. This cytokine, also known as SDF-1, is produced by thymic andbone marrow stroma (see e.g. U.S. Pat. No. 5,756,084, entitled: “Humanstromal derived factor 1α. and 1β,” issued May 26, 1998, to Honjo, etal.). CXCL12 has been reported to repel effector T-cells whilerecruiting immune-suppressive regulatory T-cells to an anatomic site.See, e.g., Poznansky et al., Nature Medicine 2000, 6:543-8. CXCL12 andits receptor CXCR4 are also reported to be an integral part ofangiogenesis.

Agents other than CXCL12 are also disclosed to repel immune cells,including, without limitation, gp120, other CXCR4 ligands, IL-8,CXCR4-binding antibodies, CXCL13, CXCR5 ligands, CXCR5-bindingantibodies, and the like.

An embodiment of the invention is a transgenic human beta cellexpressing an effective amount of a fugetactic agent, preferably CXCL12or CXCL13, so as to render the cell resistant to human immune cells. Inone embodiment, such fugetactic effective amounts of the fugetacticagent are generated by introduction of a human transgene for the agent(e.g., CXCL12, CXCL13) into the beta cell or a precursor of the betacell (e.g., a pluripotent stem cell). These human transgenic beta cellsare further characterized as expressing insulin in a hyperglycemicenvironment. As such, these cells can be used in a method for treatingdiabetes in a subject. The transgenic human beta cells used in themethods described herein may be autologous or non-autologous, e.g.,allogenic, beta cells. In one embodiment, the patient suffers from T1D.In another embodiment, the transgenic human beta cell can be modified tobe senescent (incapable of division) such that any furtherdifferentiation of these cells into cancer cells is eliminated andapoptotic induction arising due to inappropriate cell division isnegated.

An embodiment of this invention uses beta cells, e.g., autologous orallogenic beta cells, either obtained or derived from a non-diabetichuman subject or a human subject suffering from diabetes. These betacells include a functional fugetactic agent (e.g., CXCL12, CXCL13)expression vector. Such a vector is designed to express the agent in thetransgenic beta cells at a level sufficient to generate a fugetacticbuffer around the beta cells. Without wishing to be bound by theory itis contemplated that this buffer allows the beta cells to resist immunecell attack, but still express insulin as would be necessary to maintainproper blood sugar levels in a diabetic subject. The generation ofautologous beta cells from patients suffering from diabetes is known inthe art. See, for example, Egli, et al., EMBO J. 2015 Apr. 1; 34(7):841-855, which is incorporated herein by reference in its entirety.Allogeneic beta cells derived from stem cells are commerciallyavailable.

An aspect of this invention is the administration of transgenic humanbeta-cells comprising a transgene encoding a fugetactic agent (e.g.,CXCL12, CXCL13) to subjects in need thereof to modulate the levels ofinsulin and to treat diabetes in the subject. In addition, theexpression of a sufficient amount of a fugetactic agent protects againstthe risk of destruction of the transgenic beta cells by mononuclearimmune cell infiltration. The transgenic human beta cells may beautologous or allogeneic. In an embodiment of this invention, thetransgenic beta cells are autologous beta cells derived from the patientsuffering from diabetes. In another embodiment, the transgenic betacells are allogeneic human beta cells. In another embodiment, thepatient is suffering from type 1 diabetes.

Another aspect of this invention relates to transgenic human beta cellsthat are capable of expressing a fugetactic effective amount of afugetactic agent (e.g., CXCL12, CXCL13) so as to be resistant to immunedestruction. The fugetactic agent (e.g., CXCL12, CXCL13) may be anendogenous agent, i.e., an agent expressed by the subject to be treated,or an exogenous agent, e.g., an agent from a non-autologous source or amodified fugetactic agent. In one embodiment, the gene encoding thefugetactic agent in the beta cells is modified to be over-expressedcompared to the unmodified gene. Methods for modifying gene expressionare known in the art, for example, site-directed gene editing to replacethe endogenous promoter with a different promoter (e.g., a constitutivepromoter, an inducible promoter, etc.). In one embodiment, a recombinantpolynucleotide encoding the fugetactic agent is inserted into the betacells, such that the fugetactic agent is expressed from the recombinantpolynucleotide. Methods for inserting recombinant genes into a cell(transduction, transfection, etc.) are well known in the art, as aremethods for making vectors with recombinant polynucleotides forinsertion in to cells.

In some embodiments, the fugetactic agent is a modified fugetacticagent. For example, the polypeptide sequence of the fugetactic agent maybe modified to increase circulating half-life, to incorporateconservative amino acid changes, enhance binding to an extracellularmatrix, improve activity of the agent, etc. Accordingly, genes encodinga modified fugetactic agent (e.g., CXCL12 or CXCL13) can be modifiedsuch the gene has at least 95% sequence identity to the native gene andpreferably 99% sequence identity to the native gene. Likewise, the aminoacid sequence of the modified fugetactic agent (e.g., modified CXCL12 orCXCL13) has a sequence identity to the native agent of at least 95% andpreferably 99%.

In one embodiment, there is provided a human beta cell comprising avector that itself comprises a nucleic acid sequence encoding humanCXCL12 or modified CXCL12 wherein said beta cell is made resistant tohuman immune cells.

In one embodiment, the human transgenic beta cell is an autologous betacell obtained from a subject with type 1 diabetes.

In one embodiment, the human beta cell is an allogenic beta cell.

In one embodiment, the human mononuclear immune cells comprise NK cells,T cells and B cells. In one embodiment, the T cells comprise cytotoxic Tcells.

In one embodiment, the transgenic human beta cell expresses human CXCL12at a fugetactic amount.

In one embodiment, the human CXCL12 is selected from the groupconsisting of CXCL12 alpha and CXCL12 beta.

In one embodiment, the human transgenic beta cell comprises a transgenicregulatory region upstream of an endogenous CXCL12 coding region whereinsaid beta cell is resistant to human immune cells. Preferably, theendogenous CXCL12 coding region regulatory region comprises aconstitutive promoter. In some embodiments, the endogenous CXCL12 codingregion regulatory region comprises an inducible promoter.

In one embodiment, the human transgenic beta cell comprising atransgenic regulatory region upstream of an endogenous CXCL12 codingregion wherein said beta cell is resistant to human immune cells is anautologous beta cell and, preferably, one obtained from a patient withdiabetes.

In one embodiment, the human transgenic beta cell comprising atransgenic regulatory region upstream of an endogenous CXCL12 codingregion wherein said beta cell is resistant to human immune cells is anallogenic beta cell.

In one embodiment, there is provided a human transgenic beta cell thatcomprises an expressible human CXCL12 or CXCL13 gene wherein said cellexpresses a fugetactic effective amount of CXCL12 or CXC13 so as to beresistant to human immune cells.

In one embodiment, the human, transgenic beta comprises the human genefor CXCL12.

In one embodiment, the human, transgenic beta cell comprise the humangene selected from the group consisting of CXCL12 alpha and CXCL12 beta.

In one embodiment, the human, transgenic beta cell comprises the humangene for CXCL12 beta.

In one embodiment, there is provided a human, transgenic, senescent betacell that comprises an expressible human CXCL12 or CXCL13 gene whereinsaid cell expresses a fugetactic effective amount of CXCL12 or CXCl13 soas to be resistant to human immune cells and further wherein said cellis senescent.

In one embodiment, the human, transgenic, senescent beta cell comprisesan expressible human gene for CXCL12 and which is capable of insulinexpression in the presence of a hyperglycemic medium.

In one embodiment, the human, transgenic, senescent, beta cell comprisesan expressible human gene selected from the group consisting of CXCL12alpha and CXCL12 beta.

In one embodiment, the human, transgenic, beta cell comprises anexpressible human gene is CXCL12 beta.

In one embodiment, there is provided a method for producing insulin inresponse to a hyperglycemic environment which method comprisescontacting said environment with a population of human transgenic betacells as described above.

In one embodiment, the human transgenic beta cells are resistant tohuman immune cells selected from the group consisting of T cells, Bcells, NK cells, and mixtures thereof.

In one embodiment, the beta cells described herein are obtained by:

-   -   (a) obtaining a population of human progenitor cells or human        pluripotent stem cells from a human subject;    -   (b) differentiating the subject's progenitor cells or        pluripotent stem cells into beta cells; and    -   (c) introducing a nucleic acid molecule encoding the fugetactic        agent into the beta cells.

In one embodiment, the fugetactic agent is a cytokine, a chemokine, aCXCR4-binding antibody, a CXCR4 ligand, a CXCR5-binding antibody, or aCXCR5 ligand.

In one embodiment, the fugetactic agent is CXCL12, CXCL13, gp120, orIL-8.

In one aspect is provided a method for promoting survival of beta cellsin a biological sample comprising immune cells by modifying beta cellsto express a fugetactic agent at a level sufficient to inhibit or blockimmune cells from killing said beta cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of a Western blot, showing relative amounts ofCXCL12alpha, CXCL12beta, CXCL12gamma, CXCL12theta, and CXCL12delta, andCXCL14, when each is overexpressed in beta cells.

FIGS. 2A and 2B are bar graphs showing the relative amount of lactatedehydrogenase (LDH, a marker of cell lysis) released from CXCL12alpha-or CXCL12beta-expressing beta cells incubated with PBMCs at a 30:1PBMC:beta cell ratio for 24 and 48 hours.

FIG. 3 shows the level of expression of CXCL12alpha and CXCL12beta intwo sets of beta cells expressing each cytokine.

FIGS. 4A and 4B are bar graphs showing the relative amount of LDHreleased from CXCL12alpha- or CXCL12beta-expressing beta cells incubatedwith PBMCs at a 30:1 PBMC:beta cell ratio for 24 and 48 hours, with orwithout induced senescence of the beta cells (by mitomycin C treatment).

FIG. 5 shows insulin induction by hyperglycemic challenge of beta cellsexpressing CXCL12alpha or CXCL12beta, with or without treatment withmitomycin C.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides for autologous/allogeneic human beta cells thatare transgenic and comprise a transgene encoding a human fugetacticagent (e.g., CXCL12, CXCL13) or have been genetically modified toexpress or overexpress an endogenous (human) fugetactic agent (e.g.,CXCL12, CXCL13) in fugetactic amounts. In a preferred embodiment, thetransgenic beta cells described herein are further modified to besenescent. In another of its method aspects, the beta cells are modifiedor treated so as to express an effective amount of a fugetactic agent(e.g., CXCL12, CXCL13) so as to inhibit immune destruction of thetransgenic human beta cells and to produce insulin in response to ahyperglycemic environment.

Prior to disclosing this invention in further detail, the followingterms will first be defined. If a term is not defined, it has itsgenerally accepted scientific meaning as understood in the art.

The term “CXCL13” refers to all known isoforms thereof. As CXCL13 isknown to mediate certain cancer cell proliferation, it is not preferred.See, e.g., <www.ncbi.nlm.nih.gov/pmc/articles/PMC3839818/>.

The term “fugetaxis” or “fugetactic” refers to the ability of an agentto repel (or chemorepel) an eukaryotic cell with migratory capacity. Afugetactic amount of CXCL12 or CXCL13 (or other fugetactic agent)expressed by a cell is an amount sufficient to block or inhibit immunecell migration towards the cell or in some aspects repel the immunecells from the cell.

The term “human immune cell” is used interchangeably with the term“human mononuclear immune cells” and includes NK cells, T cells, and Bcells.

The term “immune cell-resistant” or “stealth to the immune system”indicates that the beta cell expresses an amount of fugetatic agent thatis sufficient to block or inhibit immune cell migration towards the cellor in some aspects repel the immune cells from the beta cell. In apreferred embodiment, such blockage or inhibition is measured by theextent of cell death after exposure of the beta cells of this inventionto human mononuclear immune cells (e.g., PBMCs). Cell death can beassessed by release of lactate dehydrogenase (LDH) from cells that haveundergone lysis. Preferably, immune cell resistant beta cells of thisinvention can be assessed by cells that evidence less than 50% of theLDH levels relative to control at a ratio of about 30:1 immune cells tobeta cells of this invention over a two day period of incubation. Morepreferably, the immune resistant beta cells evidence less than 60% ofthe LDH level relative to control; and even more preferably, less than75% of the LDH level relative to control; and most preferably, less than95% of the LDH level relative to control. The procedure for assessingLDH levels is set forth in example 2 herein.

A fugetactic agent is an agent that has fugetactic activity. Fugetacticagents may include, without limitation, CXCL12, CXCL13, gp120, IL-8,CXCR4-binding antibodies, CXCR4 ligands, CXCR5-binding antibodies, orCXCR5 ligands.

The term “effector T-cell” refers to a differentiated T-cell capable ofmounting a specific immune response by releasing cytokines.

The term “regulatory T-cell” refers to a T-cell that reduces orsuppresses the immune response of B-cells or of other T-cells to anantigen.

The terms “CXCL12” or “SDF-1 polypeptide” refer to cytokines well-knownin the art (see, for example, Table 1). In an embodiment, the termsrefer to a protein or fragment thereof that binds a CXCL12 specificantibody and that has chemotaxis or fugetaxis activity. Chemotaxis orfugetaxis activity is determined by assaying the direction of T cellmigration (e.g., toward or away from an agent of interest). See, e.g.,Poznansky et al., Nature Medicine 2000, 6:543-8; N. Papeta et al.,“Long-term survival of transplanted allogeneic cells engineered toexpress a T Cell chemorepellent,” Transplantation 2007, 83(2), 174-183.“Fugetaxis” or “Fugetactic migration” is the movement of a migratorycell away from an agent source (i.e., towards a lower concentration ofagent). It is understood that the term “CXCL12” refers to all knownisoforms thereof including the alpha, beta, gamma, delta, epsilon, phiand theta isoforms. Preferred CXCL12 isoforms are the alpha and beta.CXCL12 is known to induce angiogenesis.

The terms “type 1 diabetes” and “type 2 diabetes” refer to two majorpathophysiologies related to increased glycemia. Type 1 diabetes ischaracterized by autoimmune attack against the pancreaticinsulin-producing beta-cells whilst type 2 diabetes is associated withpoor beta-cell function and increased peripheral insulin resistance.Similar to Type 1, beta-cell death is also observed in Type 2 diabetes.Type 1 and often Type 2 diabetes requires the person to inject insulin.Type 1 diabetes is typically characterized by loss of theinsulin-producing beta-cells of the islets of Langerhans in the pancreasleading to insulin deficiency. This type of diabetes can be furtherclassified as immune-mediated or idiopathic. The majority of Type 1diabetes is of the immune-mediated nature, where beta-cell loss is dueto a T-cell mediated autoimmune attack. Type 2 diabetes is characterizedby beta-cell dysfunction in combination with insulin resistance. Thedefective responsiveness of body tissues to insulin is believed toinvolve the insulin receptor and downstream cellular signaling. Similarto Type 1 diabetes an insufficient beta cell mass is also a pathogenicfactor in many Type 2 diabetic patients. In the early stage of Type 2diabetes, hyperglycemia can be reversed by a variety of measures andmedications that improve insulin secretion and reduce glucose productionby the liver. As the disease progresses, the impairment of insulinsecretion occurs, and therapeutic replacement of insulin may sometimesbecome necessary in certain patients.

A “subject” or “patient” refers to a mammal, preferably to a humansubject.

A “subject in need thereof” or “patient in need thereof” is a subjecthaving Type 1 or Type 2 diabetes.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

The term “about” when used before a numerical designation, e.g.,temperature, time, amount, concentration, and such other, including arange, indicates approximations which may vary by (+) or (−) 10%, 5%,1%, or any subrange or subvalue there between. Other definitions appearin context throughout this disclosure.

An aspect of this invention are transgenic beta cells, e.g., humanautologous beta cells or non-autologous beta cells, e.g., allogeneicbeta cells, comprising a nucleic acid encoding a fugetactic agent (e.g.,CXCL12, CXCL13) in operable linkage with a promoter, such that thefugetactic agent (e.g., CXCL12, CXCL13) is expressed at a fugetacticlevel in the beta cell microenvironment. The promoter may be a promoterendogenous to the beta cell or heterologous to, but functional, in thebeta cell. Preferably, the nucleic acid encoding the fugetactic agent(e.g., CXCL12, CXCL13) is endogenous to the subject being treated withthe transgenic beta cells. In one embodiment, the allogeneic beta cellis derived from a non-T1D donor.

An aspect of this invention are human beta cells comprising agenetically modified endogenous human gene encoding a fugetactic agent(e.g., CXCL12, CXCL13) wherein the gene is modified to comprise aheterologous promoter in operable linkage with the fugetacticagent—encoding sequence, such that the fugetactic agent is expressedfrom the endogenous gene at a fugetactic level in the beta cellmicroenvironment. The promoter may be introduced into the beta cells tobe in operable linkage with the fugetactic agent-encoding sequence usinggenome editing techniques known in the art. It is well known that CXCL12has several isoforms including the alpha, beta, gamma, and theta. In apreferred embodiment, the isoform employed is CXCL12 beta. Thetransgenic human beta cells described herein produce insulin in responseto a hyperglycemic environment. The term “insulin” is meant to coverboth pro-insulin and insulin.

In general, this invention provides for beta cells, and preferably humanbeta cells, that express a fugetactic agent (e.g., CXCL12, CXCL13) at alevel sufficient to block or inhibit migration of immune cells (e.g.,human immune cells) to the beta cells or sufficient to repel immunecells. The terms immune cells and mononuclear cells (T-cells, B-cells,and NK cells) may be used interchangeably. The ability of a fugetacticagent (e.g., CXCL12, CXCL13) polypeptide to repel immune cells (e.g.,effector T-cells) can be assessed in vitro, using a boyden chamberassay. See, e.g., as previously described in Poznansky et al., Journalof Clinical Investigation, 109, 1101 (2002). Alternatively, theviability of transgenic human beta cells is assessed by combining suchcells with human PBMC. The rate of cell death can be evaluated bymeasuring one or more cell death markers over time. One such markercommonly used is lactate dehydrogenase (LDH) that is released duringcell necrosis.

Without wishing to be bound by any theory, Applicant contemplates thatin an aspect of this invention the amount of fugetactic agent (e.g.,CXCL12, CXCL13) produced by the transgenic beta cell is sufficient toprovide a fugetactic effect in the beta-cell microenvironment, but isnot produced in an amount sufficient to raise the systemic levels of theagent and upset the balance between the beneficial effects of the agentin one process while producing deleterious consequences in another. Inaddition, CXCL12 is known to induce angiogenesis when bound to itsreceptor CXCR4. Again, without being bound by any theory, it iscontemplated that the microenvironment of the implanted transgenic betacells expressing CXCL12 will induce an angiogenic response that enhancethe survivability of the implanted cells.

The fugetactic effective amount of a fugetactic agent (e.g., CXCL12,CXCL13) is any amount sufficient to block immune cell from killing thetransgenic beta cells. For example a fugetactic effective amount offugetactic agent (e.g., CXCL12, CXCL13) in the transgenic beta cellmicroenvironment may be at least about 100 ng/mL, and preferably atleast 100 nM. In some embodiments, the amount of fugetactic agent (e.g.,CXCL12, CXCL13) in the transgenic beta cell microenvironment is at leastabout 1000 ng/mL. For example, the following specific ranges that aresuitable for this invention: from about 100 nM to about 200 nM, fromabout 100 nM to about 300 nM, from about 100 nM to about 400 nM, fromabout 100 nM to about 500 nM, from about 100 nM to about 600 nM, fromabout 100 nM to about 700 nM, from about 100 nM to about 800 nM, fromabout 100 nM to about 900 nM, or from about 100 nM to about 1 μM.

In embodiments, the fugetactic effective amount of fugetactic agent(e.g., CXCL12, CXCL13) in the transgenic beta cell microenvironmentranges from 20 ng/mL to about 5 μg/mL. In embodiments, the fugetacticeffective amount ranges from 20 ng/mL to about 1 μg/mL. In embodiments,the amount of the fugetactic agent (e.g., CXCL12, CXCL13) in the betacell microenvironment is a fugetactic sufficient amount that ranges fromabout 100 ng/mL to about 500 ng/mL, from about 500 ng/mL to 5 μg/mL,about 800 ng/mL to about 5 μg/mL, or from about 1000 ng/mL to about 5000ng/mL. Without wishing to be bound by theory, it is contemplated thatwhen transgenic and non-transgenic beta cells are used together, thetransgenic beta cells can express sufficient amounts of the fugetacticagent such that the microenvironment creating the fugetactic effectextends to adjacent to non-transgenic beta cells. The fugetacticeffective amount of fugetactic agent (e.g., CXCL12, CXCL13) in thetransgenic beta cell microenvironment may be any value or subrangewithin the recited ranges, including endpoints.

Although mice and mouse DNA are widely used by immunologists to gaininsight into the workings of the human immune system, there aresignificant differences between humans and mice. Accordingly, thefugetactic agent (e.g., CXCL12, CXCL13) encoded by the vector ispreferably a human agent. Javier Mestas & Christopher C. W. Hughes, “OfMice and Not Men: Differences between Mouse and Human Immunology,”Journal of Immunology 2004, 172(5), 2731-2738; O. Cabrera et al., “Theunique cytoarchitecture of human pancreatic islets has implications forislet cell function,” PNAS 2006, 103(7), 2334-2339; M. Votey, “Of miceand men: how the nPOD program is changing the way researchers study type1 diabetes,” diaTribe, Network for Pancreatic Organ Donors with Diabetes(Aug. 21, 2015).

CXCL12 polypeptides are known in the art. See, e.g., Poznansky et al.,Nature Medicine 2000, 6:543-8 and US Patent Publ. No. 20170246250 bothof which are incorporated herein by reference in their entirety. Theterms CXCL12 and SDF-1 may be used interchangeably. ExemplaryCXCL12/SDF1 Isoforms are provided in Table I of US Publ. 20170246250.Exemplary CXCL12/SDF1 Isoforms are also provided in Table 1 (below):

TABLE 1 HUMAN CXCL12/SDF1 ISOFORMS Accession Accession Number NameNumber Versions Sequence SDF-1 NP_954637 NP_954637.1MNAKVVVVLV LVLTALCLSD Alpha GI:40316924 GKPVSLSYRC PCRFFESHVARANVKHLKIL NTPNCALQIV ARLKNNNRQV CIDPKLKWIQ EYLEKALNK (SEQ ID NO: 1)SDF-1 P48061 P48061.1 MNAKVVVVLV LVLTALCLSD Beta GI:1352728GKPVSLSYRC PCRFFESHVA RANVKHLKIL NTPNCALQIV ARLKNNNRQV CIDPKLKWIQEYLEKALNKR FKM (SEQ ID NO: 2) SDF-1 NP_001029058 NP_001029058.1MNAKVVVVLV LVLTALCLSD Gamma GI:76563933 GKPVSLSYRC PCRFFESHVARANVKHLKIL NTPNCALQIV ARLKNNNRQV CIDPKLKWIQ EYLEKALNKG RREEKVGKKEKIGKKKRQKK RKAAQKRKN (SEQ ID NO: 3) SDF-1 Yu et al.MNAKVVVVLV LVLTALCLSD Delta Identification GKPVSLSYRC PCRFFESHVAand expression RANVKHLKIL NTPNCALQIV of novel ARLKNNNRQV CIDPKLKWIQisoforms of EYLEKALNNL ISAAPAGKRV human stromal IAGARALHPS PPRACPTARAcell-derived LCEIRLWPPP EWSWPSPGDV factor 1. Gene (SEQ ID NO: 4)(2006) vol. 374 pp. 174-9 SDF-1 Yu et al. MNAKVVVVLV LVLTALCLSD EpsilonIdentification GKPVSLSYRC PCRFFESHVA and expressionRANVKHLKIL NTPNCALQIV of novel ARLKNNNRQV CIDPKLKWIQ isoforms ofEYLEKALNNC (SEQ ID human stromal NO: 5) cell-derived factor 1. Gene(2006) vol. 374 pp. 174-9 SDF-1 Phi Yu et al. MNAKVVVVLV LVLTALCLSDIdentification GKPVSLSYRC PCRFFESHVA and expressionRANVKHLKIL NTPNCALQIV of novel ARLKNNNRQV CIDPKLKWIQ isoforms ofEYLEKALNKI WLYGNAETSR human stromal (SEQ ID NO: 6) cell-derivedfactor 1. Gene (2006) vol. 374 pp. 174-9

In one embodiment, a CXCL12 polypeptide has at least about 85%, 90%,92%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to NP001029058 and has chemokine or fugetactic activity. In one embodiment, aCXCL12 polypeptide has at least about 85%, 90%, 92%, 95%, 96%, 97%, 98%,99%, or 100% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, and haschemokine or fugetactic activity. Such sequence identity is based on thereplacement of a first amino acid with a known conservative second aminoacid. Such conservative replacements are well established in the art andthe testing of the resulting modified CXCL12 polypeptide for itsfugetactic properties are well known in the art. See, for example,Poznansky, supra.

CXCL13 peptides are known in the art. CXCL13 is also known as Blymphocyte chemoattractant (BLC) or B cell-attracting chemokine 1(BCA-1), and these terms can be used interchangeably. For example, humanCXCL13 can be found at Accession number Q53X90. In one embodiment, aCXCL13 polypeptide has an amino acid sequence comprisingMKFISTSLLLMLLVSSLSPVQGVLEVYYTSLRCRCVQESSVFIPRRFIDRIQILPRGNGCPRKEIIVWKKNKSIVCVDPQAEWIQRMMEVLRKRSSSTLPVPVFKRKIP (SEQ ID NO: 7). In oneembodiment, a CXCL13 polypeptide has at least about 85%, 90%, 92%, 95%,96%, 97%, 98%, 99%, or 100% amino acid sequence identity to Q53X90 andhas chemokine or fugetaxis activity. In one embodiment, a CXCL13polypeptide has at least about 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%,or 100% amino acid sequence identity to SEQ ID NO: 7 and has chemokineor fugetaxis activity.

The transgenic beta cells used in the methods described herein may beautologous or non-autologous, e.g., allogenic beta cells. “Autologous”cells are cells from the same individual. “Allogeneic” cells are cellsfrom a genetically similar but not identical a donor of the samespecies. Allogenic cells useful in the methods of this invention arepreferably from a human subject. Allogenic cells useful in the methodsof this invention maybe from a relative e.g., a sibling, a cousin, aparent, or a child, or a non-relative. Criteria for selecting anallogenic donor are well known in the art see, e.g., Tatum et al.,Diabetes Metab Syndr Obes 2017: 10 73-78. Human allogeneic beta cellsare commercially available and autologous beta cells are produced by themethods described by Egli, et al., supra.

In an embodiment, the transgenic human beta cells used in the methods ofthis invention are autologous transgenic beta cells that can be preparedby deriving beta cells from multipotent progenitor cells or pluripotentstem cells obtained from the patient by methods known in the art. Thesederived beta cells may comprise (e.g., be transfected, infected, etc.with) an expression vector comprising a nucleic acid sequence encodingthe fugetactic agent (e.g., CXCL12, CXCL13).

Alternatively, the transgenic beta cells used in the methods of thisinvention may be prepared by isolating islet beta cells from the subjectin need thereof. These isolated islet beta cells may comprise (e.g., betransfected, infected, etc. with) an expression vector comprising anucleic acid sequence encoding the fugetactic agent (e.g., CXCL12,CXCL13). Alternatively, the beta cell may be genetically modified toexpress the endogenous fugetactic agent (e.g., CXCL12, CXCL13) gene suchthat it constitutively produces a fugetactic effective amount of thefugetactic agent (e.g., CXCL12, CXCL13).

In an embodiment of this invention the beta cells comprise (e.g., betransfected, infected, etc. with) an expression vector comprising anucleic acid molecule that encodes the fugetactic agent (e.g., CXCL12,CXCL13), said nucleic acid molecule being in operable linkage with apromoter suitable for expression in the beta cells. The vector mayintegrate into the genome of the beta cell or it may exist episomallyand not integrate into the genome.

The transgenic beta cells of the invention may also be prepared from anadult stem cell by isolating adult stem cells from the subject,culturing the stem cells under appropriate conditions to expand thepopulation and to induce differentiation into beta cells. The cells maybe modified to express fugetactic effective amounts of the fugetacticagent (e.g., CXCL12, CXCL13) by introducing into the cells an expressionvector encoding fugetactic amounts of the fugetactic agent (e.g.,CXCL12, CXCL13) or by editing the genome to express fugetactic amountsof the fugetactic agent (e.g., CXCL12, CXCL13). The vector may beintroduced into the stem cells prior to differentiation into beta cellsor the genome of the stem cells may be edited to contain theheterologous promoter. Alternatively, the vector may be introduced intothe resulting beta cells or the genome of the resulting beta cells maybe edited to contain the heterologous promoter.

The transgenic beta cells of the invention may also be prepared bygenerating induced pluripotent stem (iPS) cells from somatic cells,e.g., beta cells, fibroblasts or keratinocytes, of a subject; treatingthe iPS cells to induce differentiation into beta cells; and introducinginto the differentiated beta cells an expression vector comprising anucleic acid sequence encoding the fugetactic agent (e.g., CXCL12,CXCL13).

The transgenic beta cells of the invention may also be prepared bypreparing induced pluripotent stem (iPS) cells generated from somaticcells of a subject; introducing into the iPS cells an expression vectorcomprising a nucleic acid sequence encoding the fugetactic agent (e.g.,CXCL12, CXCL13); and treating the iPS cells, before or afterintroduction of the transgene, to induce differentiation into betacells.

The transgenic beta cells of this invention may also be generated byobtaining progenitor cells or progenitor-like cells, e.g., pancreaticβ-cell progenitors, introducing a vector comprising a nucleic acidsequence encoding the fugetactic agent (e.g., CXCL12, CXCL13) into thecells, and treating the cells either before or after introducing thevector to induce differentiation into beta cells, or insulin releasingcells responsive to glucose levels in the body, by methods known in theart, see e.g., Millman et al. Nature Communications (10 May 2016) page1-8); Baek et al. Curr Stem Cell Rep (2016) 2:52-61; Russ et al., EMBO.J. 34, 1759-1772 (2015); and, Qadir et al., Cell Reports 22, 2408-2420(Feb. 27, 2018). The progenitor cell and progenitor-like cells may beautologous or non-autologous, e.g., allogeneic, to the subject treatedwith the transgenic cells. Insulin-producing cells responsive to glucoselevels in the body, (see e.g., Qadir et al., supra), may be geneticallymodified as described herein to express fugetactic levels of thefugetactic agent (e.g., CXCL12, CXCL13) and are also an embodiment ofthis invention. Such genetically modified insulin producing cells canlikewise be used in the methods of this invention to treat diabetes asdescribed herein.

Any suitable somatic cell from a subject may be reprogrammed into an iPScell by methods known in the art, see e.g., Pagliuca and Melton (2013)How to make a functional β-cell, Development (3013) 140(12); 2472-2483;Yu et al. (2007). Induced pluripotent stem cell lines derived from humansomatic cells. Science 318, 1917-1920; Takahashi and Yamanaka, 2006,Cell 126(4):663-676; Wernig et al., 2007, Nature 448:7151; Okita et al.,2007 Nature 448:7151; Maherali et al., 2007 Cell Stem Cell 1:55-70;Lowry et al., 2008 PNAS 105:2883-2888; Park et al., 2008 Nature451:141-146; Takahashi et al., 2007 Cell 131, 861-872; U.S. Pat. Nos.8,546,140; 7,033,831 and; 8,268,620. The iPS cells may be differentiatedinto beta cells using methods known in the art, see e.g. US patentpublication no. 20170081641 and US patent publication no 20130164787,and Millette and Georgia, “Gene Editing and Human Pluripotent StemCells: Tools for advancing Diabetes Disease Modeling and Beta CellDevelopment”, Current Diabetes Reports November 2017, 17: 116; US patentapplication no. 20130273651; Shi, Y., et al. Stem Cells., 25: 656-662(2005); or Tateishi, K., et al., J Biol Chem., 283: 31601-31607 (2008).

Preferably the fugetactic agent-encoding sequence is in operable linkagewith a regulatory region that is suitable for expression in a beta cell.Suitable regulatory regions are known in the art, and include promoterssuch as, e.g., mammalian promoters including, e.g., hypoxanthinephosphoribosyl transferase (HPTR), adenosine deaminase, pyruvate kinase,β-actin promoter, muscle creatine kinase promoter, and human elongationfactor promoter (EF1α), a GAPDH promoter, an actin promoter, and anubiquitin promoter and viral promoters including SV40 early promoter,SV40 late promoter, metallothionein promoter, murine mammary tumor viruspromoter, Rous sarcoma virus promoter, polyhedrin promoter, humanimmunodeficiency virus (HIV) promoters, cytomegalovirus (CMV) promoters,adenoviral promoters, adeno-associated viral promoters, or the thymidinekinase promoter of herpes simplex virus. Other relevant promoters, e.g.,viral and eukaryotic promoters, are also well known in the art (seee.g., in Sambrook and Russell (Molecular Cloning: a laboratory manual,Cold Spring Harbor Laboratory Press). The regulatory region in operablelinkage with the fugetactic agent-encoding sequence may be anyconstitutive promoter suitable for expression in the subject's cells.

The transgenic cells expressing the fugetactic agent (e.g., CXCL12,CXCL13) of this invention, whether autologous or non-autologous, e.g.,allogeneic, may be administered to a subject in need thereof by anymeans known in the art for administering beta cells. The transgeniccells of this invention may be administered in an amount sufficient toprovide levels of insulin able to alleviate at least some of thesymptoms associated with low levels of insulin.

Another aspect of the invention is a method of treating diabetes in asubject in need thereof, comprising the steps of: (a) obtaining orderiving beta cells or insulin-producing beta-like cells, from thesubject; (b) introducing a suitable expression vector encoding thefugetactic agent (e.g., CXCL12, CXCL13) into the cells to formautologous transgenic cells expressing the introduced the fugetacticagent (e.g., CXCL12, CXCL13); and (c) transplanting the autologoustransgenic cells into the subject.

Many vectors useful for transferring exogenous genes into mammaliancells, e.g., beta cells, including vectors that integrate into thegenome and vectors that do not integrate into the genome but exist asepisomes, and methods for introducing such vectors into cells areavailable and known in the art. For example, retroviral vectors,lentiviral vectors, adenoviral vectors, adeno-associated (AAV)-basedvectors and EBV-based vectors may be used. See, e.g., US 20110280842,Narayanavari and Izsvak, Cell Gene Therapy Insights 2017; 3(2), 131-158;Hardee et al., Genes 2107, 8, 65; Tipanee et al., Bioscience Reports(2017) 37, and Chira et al. Oncotarget, Vo. 6, No. 31, pages30675-30703.

Another aspect of the invention is a method for promoting survival ofbeta cells in a biological sample comprising immune cells comprisingintroducing an expression vector encoding the fugetactic agent (e.g.,CXCL12, CXCL13) into the beta cells, or by editing the genome of thebeta cells such that the beta cells express fugetactic amounts of thefugetactic agent (e.g., CXCL12, CXCL13). In an aspect of this inventionthe fugetactic agent (e.g., CXCL12, CXCL13) is expressed by the betacells at a level sufficient to block or inhibit migration of immunecells, e.g. T-cells, B-cells, and/or NK cells, to the beta cells. In anaspect of this invention the fugetactic agent (e.g., CXCL12, CXCL13) isexpressed by the beta cells at a level sufficient to repel the immunecells from the beta cells. In an aspect of this invention thegenetically modified beta cells are in a subject, e.g., a human subjecthaving Type 1 or Type 2 diabetes. In one embodiment, the beta cells areautologous beta cells of the subject.

Methods for the delivery of viral vectors and non-viral vectors tomammalian cells are well known in the art and include, e.g.,lipofection, microinjection, ballistics, virosomes, liposomes,immunoliposomes, polycation or lipid-nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Lipofectionreagents are sold commercially (e.g., Transfectam™ and Lipofectin™).Cationic and neutral lipids suitable for efficient receptor-recognitionlipofection of polynucleotides are known. Nucleic acid can be deliveredto cells (ex vivo administration) or to target tissues (in vivoadministration). The preparation of lipid:nucleic acid complexes,including targeted liposomes such as immunolipid complexes, is wellknown to those of skill in the art. Recombination mediated systems canbe used to introduce the vectors into the cells. Such recombinationmethods include, e.g., use of site specific recombinases like Cre, Flpor PHIC31 (see e.g. Oumard et al., Cytotechnology (2006) 50: 93-108)which can mediate directed insertion of transgenes.

Vectors suitable for use in this invention include expression vectorscomprising a nucleic acid encoding a fugetactic agent (e.g., CXCL12,CXCL13) in operable linkage with a promoter to direct transcription.Suitable promoters are well known in the art and described, e.g., inSambrook and Russell (Molecular Cloning: a laboratory manual, ColdSpring Harbor Laboratory Press). The promoter used to direct expressionof the fugetactic agent (e.g., CXCL12, CXCL13) may be, e.g., example,SV40 early promoter, SV40 late promoter, metallothionein promoter,murine mammary tumor virus promoter, Rous sarcoma virus promoter, orother promoters shown to be effective for expression in mammalian cells.

Vectors useful in the methods of this invention include, e.g., SV40vectors, papilloma virus vectors, Epstein-Barr virus vectors, retroviralvectors, and lentiviral vectors.

The vectors used in this invention may comprise regulatory elements fromeukaryotic viruses, e.g., SV40, papilloma virus, and Epstein-Barr virus,including e.g., signals for efficient polyadenylation of the transcript,transcriptional termination, ribosome binding, and/or translationtermination. Additional elements of the vectors may include, e.g.,enhancers, and heterologous spliced intronic signals.

In an embodiment of this invention, the genome of the beta cell may begenetically modified to increase the expression levels of an endogenousfugetactic agent (e.g., CXCL12, CXCL13) gene. Such increased expressionmay be achieved by introducing a heterologous promoter in operablelinkage with the endogenous fugetactic agent (e.g., CXCL12, CXCL13) geneor by altering the endogenous fugetactic agent (e.g., CXCL12, CXCL13)promoter such that the beta cell expresses a fugetactic level offugetactic agent (e.g., CXCL12, CXCL13). Such increased expression maybe achieved by introducing a promoter into the genome of the beta cellsuch that it is in operable linkage with the endogenous fugetacticagent-encoding sequence and thereby expresses or overexpresses thefugetactic agent in a fugetactic amount.

Gene editing technologies for modifying the genome are well known in theart and include e.g., CRISPR/CAS 9, Piggybac, Sleeping Beauty genomeediting systems, (see for example, Zhang et al. Molecular TherapyNucleic Acids, Vol 9, December 2017, page 230-241; systems (see e.g.,Cong et al., Science. 2013; 339(6121): 819-23; Mali et al., Science.2013; 339(6121): 823-6; Gonzalez et al., Cell Stem Cell. 2014; 15(2):215-26); He et al., Nucleic Acids Res. 2016; 44(9); Hsu et al., Cell.2014; 157(6): 1262-78), zinc finger nuclease-based systems (see e.g.,Porteus and Carroll, Nat Biotechnol. 2005; 23(8): 967-73; Urnov et al.,Nat Rev Genet. 2010; 11(9): 636-46), TALEN-based systems (transcriptionactivator-like effector nucleases)(see e.g., Cermak et al., NucleicAcids Res. 2011; 39(12); Hockemeyer et al., Nat Biotechnol. 2011; 29(8):731-4; Joung and Sander J D, Nat Rev Mol Cell Biol. 2013; 14(1): 49-55;Miller et al., Nat Biotechnol. 2011; 29(2): 143-8, and Reyon et al., NatBiotechnol. 2012; 30(5): 460-5).

In one embodiment, the transgenic beta cells described herein aretreated with an agent that renders the cells viable and capable ofcontrolling blood sugar in a patient but unable to replicate (i.e.,induced cellular senescence). One such agent is Mitomycin C that is aknown DNA cross-linking agent. Upon treatment, the DNA in these cells iscross-linked thereby rendering impossible the formation of singlestranded DNA necessary for replication. Such a treatment prevents thecells, especially those generated from stem cells, from dividing suchthat if the cell morphs into a cancer cell it cannot divide. Other knownagents capable of inducing cellular senescence include those recited byPetrova, et al., “Small Molecule Compounds that Induce CellularSenescence” Aging Cell, 15(6):999-1017 (2016) which reference isincorporated herein in its entirety. Such agents include, by way ofexample only, agents that cause telomere dysfunction due toreplication-associated telomere shortening, subcytoxic stresses such asexposure to UV, gamma irradiation, hydrogen peroxide, and hypoxia. Thespecific means by which the beta cells of this invention are renderednon-replicative is not critical provided that these cells can beimplanted without risk of cellular division. Studies show adult humanpancreas has very little beta cell turnover, suggesting that limitingthe ability of the cells to divide will have little to no effect oninsulin production by implanted cells. See, e.g., Perl et al., TheJournal of Clinical Endocrinology & Metabolism, Volume 95, Issue 10, 1Oct. 2010, Pages E234-E239.

Another aspect of the invention is a method of modulating the levels ofinsulin in a subject, comprising administering to the subject in needthereof the beta cells of this invention wherein the beta cells expressinsulin and produce a fugetactic agent (e.g., CXCL12, CXCL13) in afugetactic amount. The beta cells may be autologous beta cells ornon-autologous beta cells, e.g. allogeneic beta cells, and may harbor avector expressing the fugetactic agent, which vector may be integratedinto the beta cell genome or exist episomally. In an embodiment of thisinvention the transgenic beta cells may be a genetically modified tooverexpress endogenous fugetactic agent (e.g., CXCL12, CXCL13) at afugetactic level.

Methods of introducing the transgenic beta cells described herein intoindividuals are well known to those of skills in the art and include,but are not limited to, injection, intravenous, intraportal, orparenteral administration. Single, multiple, continuous or intermittentadministration can be effected. See e.g., Schuetz and Markmann, CurrTransplant Rep. 2016 September; 3(3): 254-263.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of media and agentsfor pharmaceutically active substances, including cells, is well knownin the art. A typical pharmaceutical composition for intravenousinfusion of beta cells could be made up to contain 250 ml of sterileRinger's solution, and 100 mg of the combination. Actual methods forpreparing parenterally administrable compounds will be known or apparentto those skilled in the art and are described in more detail in forexample, Remington's Pharmaceutical Science, 17th ed., Mack PublishingCompany, Easton, Pa. (1985), and the 18th and 19th editions thereof,which are incorporated herein by reference.

The transgenic and genetically modified beta cells of the invention canbe introduced into any of several different sites well known in the art,including but not limited to the pancreas, the abdominal cavity, thekidney, the liver, the portal vein or the spleen of the subject.

In addition, in order to avoid any possible transformation of thetransgenic beta cells into cancer cells that could result in thepossibility of the patient developing a tumor, the transgenic beta cellscan be rendered senescent by contacting with known agents such asMitomycin C, or exposure to subtoxic stress from ionizing radiation,hypoxia, hydrogen peroxide, etc. Cells derived from pluripotent stemcells typically undergo apoptosis during inappropriate cell division ordue to immune cell clearance. The senescent transgenic beta cellsdescribed herein are incapable of division thereby eliminating apoptotictriggers arising during cellular division. In addition, the senescenttransgenic beta cells described herein are immune cell resistant therebyproviding protection against apoptosis induction due to immune cellclearance. Accordingly, it is contemplated that the transgenic betacells described herein will have a longer lifespan to a significantlylonger lifespan than non-senescent transgenic beta cells.

The transgenic and preferably senescent modified beta cells may betransplanted into the subject via a graft. An ideal beta celltransplantation site would be one that supports the implantation,long-term function and survival of grafted cells in the subject and iseasily accessible for maximal patient safety. Sites for implantationinclude the liver, intestinal, subdermal, and pancreatic sites.

The following abbreviations used herein have the following meanings andif abbreviations are not defined, they have their generally acceptedscientific meaning. Amino acids are recited herein using theirestablished one letter abbreviations.

FLAG = DYKDDDDK protein tag (SEQ ID NO: 10) g/L = grams per liter HRP =horseradish peroxidase LDH = lactate dehydrogenase iBLOT = Semi-dryprotein transfer device (Invitrogen) MES =2-(N-morpholino)ethanesulfonic acid mL = milliliter N/A = not applicablenM = nanomolar PBMC = peripheral blood mononuclear cells PBS = phosphatebuffered saline TMB = 3,3′,5,5′-Tetramethylbenzidine μL = microliters μg= micrograms x g = times gravity

EXAMPLES Example 1: Model Cells Used to Assess Expression Levels ofCXCL12-a and -b Isoforms

HEK293 cells were transfected with 2 different isoforms of CXCL12 (alphaand beta) using commercially available plasmids for each isoform(plasmids available from GenScript). Transfected cells were selectedwith 250 ug/mL of G418 (commercially available from ThermoFisher) and astable pool for each isoform was created. Cells were allowed tocondition a suitable medium for 3 days. Conditioned medium from thetransfected HEK293 cells expressing CXCL12 alpha and CXCL12 beta werediluted 1:1 with assay dilution buffer. Two separate pools wereestablished for each isoform and then the concentration of each isoformin solution were obtained by absorption using a standardizedconcentration curve. This experiment was repeated twice and the resultsare as follows:

CXCL12 alpha CXCL12 beta 1. 310 nM 1410 nM 2. 274 nM 1330 nM

The above results evidence that transgenic model cells express CXCL12beta at significantly higher levels than transgenic model cells thatexpress CXCL12 alpha.

Example 2—Model Cells Used to Assess Expression Levels of Other Isoformsof CXCL12

HEK293 cells were transfected with 5 different isoforms of CXCL12 (alphaand beta) using commercially available plasmids for each isoform(plasmids available from GenScript). Transfected cells were selectedwith 250 ug/mL of G418 (commercially available from ThermoFisher) and astable pool for each isoform was created. Cells were allowed tocondition in a suitable medium for 3 days. The conditioned medium wasseparated in a 4-8% NuPage gel (commercially available fromThermoFisher) with MES buffer and transferred to nitrocellulose (iBLOT).

Expression levels were detected with HRP labeled, anti-FLAG tagantibody/TMB chromogen (available from GenScript) on a Western Blot, asshown in FIG. 1. The results evidenced that the gamma, delta and thetaisoforms of CXCL12 had greater concentrations than the alpha or betaisoforms.

Example 3: Preparation of Transgenic Beta Cells

Pancreatic beta cells derived from human induced pluripotent stem cellswere purchased from Takara Bio USA, Inc. (Mountain View, Calif.) andcultured according to provided instructions.

Cells were transduced with lentiviral vectors (pLenti-C-Myc-DDK, OriGeneTechnologies, Rockville, Md.) containing a human CXCL12 isotype(CXCL12a/SDF-1alpha or CXCL12b/SDF-1beta) or control. The lentiviralvectors were used at a ratio of about 10:1 per beta cell. The sequences,including the tag (underlined) are provided below. Concentration of theCXCL12 isotype was determined by ELISA (RayBioTech, Norcross, Ga.)(Table 1).

CXCL12a (aka SDF1a) Accession No. NM_199168 SEQ. ID NO.: 9ATGAACGCCAAGGTCGTGGTCGTGCTGGTCCTCGTGCTGACCGCGCTCTGCCTCAGCGACGGGAAGCCCGTCAGCCTGAGCTACAGATGCCCATGCCGATTCTTCGAAAGCCATGTTGCCAGAGCCAACGTCAAGCATCTCAAAATTCTCAACACTCCAAACTGTGCCCTTCAGATTGTAGCCCGGCTGAAGAACAACAACAGACAAGTGTGCATTGACCCGAAGCTAAAGTGGATTCAGGAGTACCTGGAGAAAGCTTTAAACAAGACGCGTACGCGGCCGCTCGAGCAGAAACTCATCTCAGAAGAGGATCTGGCAGCAAATGATATCCTGGATTACAAGGATGACGA CGATAAGGTTTAACXCL12b (aka SDF1b) Accession No. NM_000609 SEQ. ID NO.: 8ATGAACGCCAAGGTCGTGGTCGTGCTGGTCCTCGTGCTGACCGCGCTCTGCCTCAGCGACGGGAAGCCCGTCAGCCTGAGCTACAGATGCCCATGCCGATTCTTCGAAAGCCATGTTGCCAGAGCCAACGTCAAGCATCTCAAAATTCTCAACACTCCAAACTGTGCCCTTCAGATTGTAGCCCGGCTGAAGAACAACAACAGACAAGTGTGCATTGACCCGAAGCTAAAGTGGATTCAGGAGTACCTGGAGAAAGCTTTAAACAAGAGGTTCAAGATGACGCGTACGCGGCCGCTCGAGCAGAAACTCATCTCAGAAGAGGATCTGGCAGCAAATGATATCCTGGATTACAAGGATGACGACGATAAGGTTTAA

Example 4: Transgenic Beta Cells Repel PBMCs

The transgenic beta cells from Example 3 were contacted with humanperipheral blood mononuclear cells (PBMCs, Innovative Research, Novi,Mich.) at a ratio of 30:1 (PBMCs to beta cell). Briefly, PBMCs wereresuspended in beta full culture medium, counted and adjusted to allowfor a 30:1 PBMC:beta cell ratio with addition of 100 uL of PBMCs (tominimize dilution of the expressed CXCL12). Final volume was 1.1 mL.Background controls of beta cells without PBMCs and PBMCs without betacells were also created. Immediately 150 uL of medium was removed fromeach sample and centrifuged at 1200×g for 10 minutes. Supernatant wasremoved and stored at 4° C. (time zero). Cells were returned to theincubator and sampled in a similar way to the time zero sample at both24 and 48 hours later.

Release of LDH was tested at 24 and 48 hours after contact using PierceLDH Cytotoxicity Assay Kit (Thermo Scientific) according tomanufacturer's instructions. Increased LDH is an indicator ofcytotoxicity (cell lysis).

Data (background subtracted) from a representative experiment areprovided in Table 2 and FIG. 2A. Data from a second representativeexperiment are provided in FIG. 2B.

TABLE 2 LDH and CXCL12 Levels Cytokine LDH - 24 hr LDH - 48 hr CytokineConc. Control 170 375 N/A SDF1a 52 75 ~100 nM SDF1b 4 7 ~400 nM

These data indicate that expression of CXCL12 by beta islet cellsprotects the beta islet cells from immune cell attack thereby renderingthem resistant. Beta cells expressing SDF1b/CXCL12b, which was expressedat a higher level than SDF1a/CXCL12a in this experiment, showsessentially no cytotoxicity in the presence of PBMCs.

Example 5: Alternative Preparation of Transgenic Beta Cells

Beta cells are isolated from a subject having type 1 diabetes aretransfected or infected in vitro with a retroviral expression vectorencoding CXCL12 or a control retroviral vector that does not encodeCXCL12. Transgenic beta cells harboring the retroviral vector encodingCXCL12 are assayed for expression of fugetactic amounts of CXCL12 usinga Boyden chamber assay as previously described in Poznansky et al.,Journal of Clinical Investigation, 109, 1101 (2002). It is expected thattransgenic beta cells expressing at least 100 nM CXCL12 will repelimmune cells in this assay.

Example 6: Effect of Forced Senescence of Transgenic Beta Cells onTransgenic Cytokine Expression

Beta cells were prepared as described in Example 3. Expression levels ofSDF1a/CXCL12a and SDF1b/CXCL12b were assayed by ELISA before Mitomycin C(available from Santa Cruz Biotechnology) treatment to determinebaseline expression (“Before”). Medium was replaced with fresh mediumcontaining 10 ug/mL Mitomycin C—an agent known to induce senescence.Cells were returned to the incubator for 2 hours. The mitomycin Ccontaining medium was removed by gentle pipetting. The cells were washedwith PBS twice. After the second wash, the cells were fed fresh completemedium. SDF1a/CXCL12a or SDF1b/CXCL12b expression was determined byELISA assay.

Data from two representative experiments are shown in Table 3 and FIG.3A. SDF1a/CXCL12a and SDF1b/CXCL12b expression is not affected by forcedsenescence of the transgenic beta cells.

TABLE 3 CXCL12a and -b levels before and after Mitomycin C treatmentCytokine Before Mitomycin C After Mitomycin C CXCL12a  97.5 nM 100.5 nMPool 12 CXCL12a  98.2 nM  99.4 nM Pool 22 CXCL12b 806.2 nM 789.3 nM Pool12 CXCL12b 757.1 nM 788.0 nM Pool 22

Example 7: Effect of Forced Senescence of Transgenic Beta Cella on PBMCChallenge

Beta cells were prepared as described in Example 3. Cells were treatedwith Mitomycin C or control as described in Example 4. Cells werecontacted with PBMCs as described in Example 2.

Data from two representative experiments are shown in FIGS. 4A and 4B.LDH levels are not affected by forced senescence of the transgenic betacells.

Example 8: Effect of Forced Senescence of Transgenic Beta Cells onInsulin Production

Beta cells were prepared as described in Example 3. Cells were treatedwith Mitomycin C or control as described in Example 4.

Full growth medium was replaced with 1 mL of Medium 2 and maintained onMedium 2 for 2 days with medium replacement every 24 hours. On day 3,the beta cells were challenged with the hyperglycemic medium (4.5 g/Lglucose). Samples of conditioned media were taken 24 hours followinghyperglycemic challenge and insulin expression was measured by sandwichELISA.

Data from two representative experiments are shown in Table 4 and FIG.5. The results evidence that transgenic beta cells and transgenicsenescent beta cells produced substantially equal amounts of insulin asthe control beta cells in response to hyperglycemic challenge.

TABLE 4 Insulin Expression in response to hyperglycemic challenge SampleBefore Mytomycin C After Mytomycin C CXCL12a Pool 12 170.9 mIU/ml 170.0mIU/ml CXCL12a Pool 22 172.3 mIU/ml 169.5 mIU/ml CXCL12b Pool 12 169.6mIU/ml 167.4 mIU/ml CXCL12b Pool 22 169.6 mIU/ml 168.1 mIU/ml Nocytokine Pool 12 168.3 mIU/ml 168.2 mIU/ml No cytokine Pool 22 168.9mIU/ml 167.9 mIU/ml No hyperglycemic Pool 12  6.04 mIU/ml  5.97 mIU/mlNo hyperglycemic Pool 22  6.70 mIU/ml  6.36 mIU/ml

Example 9: In Vivo Evaluation of Transgenic Beta Cells

Humanized mice having a humanized immune system, see e.g., N. Walsh,“Humanized mouse models of clinical disease,” Annu Rev Pathol 2017, 12,187-215; E. Yoshihara et al., are administered either the transgenichuman beta cells expressing fugetactic amounts of CXCL12 or the controltransgenic human beta cells and the production of insulin and survivalof the transgenic beta cells in the mice are assayed at various timepoints after the initial administration. It is contemplated that thetransgenic human beta cells expressing fugetactic amounts of CXCL12 willsurvive for longer periods than the control transgenic human beta cells.It is also contemplated that the mice receiving the transgenic betacells expressing fugetactic amounts of CXCL12 will also have higheramounts of human insulin than mice receiving the control transgenichuman beta cells and the higher levels of human insulin will persist forlonger periods of time as compared to the levels in mice administeredthe control transgenic human beta cells.

Humanized mice having a humanized immune system, see e.g., N. Walsh,“Humanized mouse models of clinical disease,” Annu Rev Pathol 2017, 12,187-215; E. Yoshihara et al., are administered either geneticallymodified human beta cells overexpressing CXCL12 from an endogenousCXCL12 gene, or control human beta cells and the production of humaninsulin and survival of the beta cells in the mice are assayed atvarious time points after the initial administration. It is contemplatedthat the genetically modified human beta cells overexpressing CXCL12will survive for longer periods than the control human beta cells thatwere not genetically modified to overexpress CXCL12. It is alsocontemplated that mice receiving the genetically modified human betacells overexpressing CXCL12 will also have higher amounts of humaninsulin than mice receiving the control human beta cells and the higherlevels of human insulin will persist for longer periods of time ascompared to the levels in mice administered the control human betacells.

Optionally, the cells are treated with an agent that cross-links the DNAwithin the cell to prevent cell division (e.g., Mitomycin C).

The foregoing description has been set forth merely to illustrate theinvention and is not meant to be limiting. Since modifications of thedescribed embodiments incorporating the spirit and the substance of theinvention may occur to persons skilled in the art, the invention shouldbe construed broadly to include all variations within the scope of theclaims and equivalents thereof.

The invention claimed is:
 1. A population of isolated geneticallyengineered human beta cells comprising a nucleic acid encoding forexogenous or heterologous expression of a human CXCL12 protein betaisoform such that said population of said cells is characterized asbeing resistant to cell death when in the presence of human immune cellsin vitro as compared to a population of unengineered human beta cellsobtained from a human stem cell that has been differentiated ex vivointo human beta cells, wherein said characterization is based on therelative amounts of lipase dehydrogenase (LDH) measured after assayingsaid population of engineered cells for 24 hours in the presence ofhuman peripheral blood mononuclear cells (PBMCs) at a ratio of 1:30genetically engineered beta cells to PBMCs, wherein the amount of LDH isdecreased by at least 95% for said population of engineered human betacells compared to that of a population of unengineered human beta cells,wherein the nucleic acid is operably linked to an exogenous promoter,wherein the population of genetically engineered human beta cells isinsulin producing and has been exposed to a senescence inducing agent,and wherein the population of genetically engineered human beta cells isobtained from a human stem cell that has been differentiated ex vivointo a human beta cell and said population of genetically engineeredhuman beta cells is allogeneic or autologous.
 2. The population ofisolated genetically engineered human beta cells of claim 1, wherein thehuman CXCL12 protein isoform comprises SEQ ID NO:
 2. 3. The populationof isolated genetically engineered human beta cells of claim 2, whereinsaid population of isolated genetically engineered human beta cells isan induced pluripotent stem (iPS) cell that is an allogeneic iPSdifferentiated into a human beta cell.
 4. The population of claim 1,wherein said characterization is based on the relative amounts of LDHmeasured after assaying said population of engineered cells for 48 hoursin the presence of PMBCs.
 5. The population of claim 1, wherein thenucleic acid is heterologous.
 6. The population of claim 1, wherein saidpopulation of genetically engineered human beta cells is autologous. 7.The population of claim 1, wherein said population of geneticallyengineered human beta cells is allogeneic.